Method of site-specific nucleic acid cleavage

ABSTRACT

A method of cleaving a target nucleic acid molecule is disclosed. A cleavage structure is formed that comprises the target nucleic acid and a pilot nucleic acid. A first region of the target nucleic acid is annealed to the pilot nucleic acid to form a duplex structure. A second region of the target nucleic acid contiguous to the duplex is not annealed to the pilot nucleic acid, thus forming a junction site between the duplex region and the non-annealed region. The cleavage structure is exposed to a cleavage agent capable of preferentially cleaving the cleavage structure at a target site in a manner independent of the sequence of the cleavage structure. The cleavage structure and the cleavage agent are incubated under conditions wherein cleavage can occur.

This is a continuation of application Ser. No. 07/986,330, filed Dec. 7,1992, U.S. Pat. No. 5,422,253.

FIELD OF THE INVENTION

The field of the present invention is molecular biological techniquesfor manipulating nucleic acid molecules. In particular, the field of thepresent invention is use of a 5' exonuclease activity of a DNApolymerase to cleave a nucleic acid molecule.

BACKGROUND

Cleavage of Nucleic Acid

Many techniques of molecular biology depend on the ability to cleave DNAor RNA molecules specifically at defined locations. Restriction enzymescleave double-stranded DNA at specific sequences that are usuallypalindromic and 4-6 base pairs in length. Several hundred restrictionenzymes have been discovered, most of which only cleave double-strandedDNA molecules. Several restriction enzymes have been shown to have theability to cleave single-stranded DNA, albeit with reduced efficiency,at sites that bear the sequence that is normally recognized indouble-stranded DNA. While the large number of different restrictionenzymes cleave double-stranded DNA at a variety of sites, these enzymescleave only at sites whose sequences conform to the substrate sequencespecificity of the enzyme, and do not cleave at all points that might bedesired by the investigator. Therefore, restriction enzymes limit acleavage reaction to both a specific nucleotide sequence and to the useof double-stranded DNA.

Class IIs restriction enzymes, such as Fok 1, cleave DNA at a site otherthan their recognition sequences. Fok I can be directed to cleavesingle-stranded DNA at selected sites through the use of adaptoroligonucleotides that direct it to the DNA (Podhajska et al., Gene, 40,175-182; 1985). The adaptor must contain two regions--one that serves asthe recognition site for the enzyme and another that hybridizes to thesingle-stranded DNA. The specificity of the binding of the adaptor toits target may be relatively low due to the ability of the enzyme totolerate mismatched base pairs and the need to incubate the reaction attemperatures below 40° C. Only DNA has been shown to be a substrate forcleavage in this system.

Ribozymes, RNA molecules that possess self-catalytic activity, can betargeted to cleave nucleic acid. However, the specific target cleavagesites have a sequence requirement (Symons, R. Ann. Rev. of Biochem.,61:641-671, 1992).

Other methods of cleaving nucleic acids include the use of non-specificnucleases. A nuclease is an enzyme that cleaves nucleic acids.Endonucleases, such as the restriction endonucleases discussed above,cleave nucleic acids by hydrolysis of internal phosphodiester bonds.Although restriction endonucleases cleave only at specific nucleotidesequences, other endonucleases, such as Mung Bean nuclease, are notsequence-specific. In contrast, exonucleases cleave nucleic acid chainsfrom the ends. An example of a structure-specific nuclease is snakevenom phosphodiesterase I which is a nuclease that degradessingle-stranded nucleic acids. Non-sequence-specific nucleases, eitherexonucleases or endonucleases, cannot he used directly to cleave nucleicacid molecules in a sequence specific manner.

RNA can be cleaved at specific sites through hybridization of adaptormolecules that serve as sequence-specific recognition sites for RNasessuch as RNaseP. (Li, et al., Proc. Natl. Acad. Sci., 89:3185-3189,1992.)

There is a need in the art of molecular biology techniques for a methodto cleave nucleic acids at any specific sequence that is not limited tosequences recognized by restriction endonucleases.

DNA Polymerase

DNA polymerases (DNAPs) catalyze the synthesis of a DNA chain.Additionally, many DNAPs are known to have nuclease activity.

Some DNAPs are known to remove nucleotides from the 5' and 3' ends ofDNA chains (Kornberg, et al., DNA Replication, 2d ed., W. H. Freeman andCo., publishers, 1992). These activities are usually termed "5'exonuclease" and "3' exonuclease", respectively. For example, the 5'exonuclease activity located in the N-terminal domain of several DNAPsparticipates in removal of RNA primers during lagging strand synthesisduring replication and the removal of damaged nucleotides during repair.Some DNAPs, such as one isolated from E. coli (DNAPEcl), also have a 3'exonuclease activity responsible for proof-reading during synthesis(Kornberg, supra).

A DNAP isolated from Thermus aquaticus, called Taq DNA polymerase(DNAPTaq), has a 5' exonuclease activity, but lacks a functional 3'exonucleolytic domain (Lawyer, et al., J. Biol. Chem. Sci., 12:288,1987). Derivatives of DNAPEcl and DNAPTaq, respectively called theKlenow (DNAPKln) and Stoffel (DNAPStf) fragments, lack 5' exonucleasedomains as a result of enzymatic or genetic manipulations (Brutlag, etal., Biochem. Biophys. Res. Commun., 37:982, 1969; Erlich, et al.,Science, 252:1643, 1991; Setlow, et al., J. Biol. Chem., 247:232, 1972).The 5' exonuclease activity of DNAPTaq is reported to require concurrentsynthesis (D. H. Gelfand, PCR Technology; Principles and ApplicationsFor DNA Amplification, Henry A. Erlich, ed. Stockton Press, 17, 1989).Although mononucleotides predominate among the digestion products of the5' exonucleases of DNAPTaq and DNAPEcl, short oligonucleotides (≦12nucleotides) can also be observed, implying that these so-called 5'exonucleases can function endonucleolytically (Setlow, supra; Holland,et al. Proc. Natl. Acad. Sci. USA., 88:7276, 1991). Thus, we prefer tocall these activities "5' nucleases".

SUMMARY OF THE INVENTION

The present invention is a method of cleaving a single-stranded nucleicacid molecule. We refer to this single stranded nucleic acid molecule asthe "target" molecule.

First, a cleavage structure is formed that comprises the target nucleicacid and a pilot nucleic acid. A first region of the target nucleic acidis annealed to the pilot nucleic acid to form a duplex structure. Asecond region of the target nucleic acid contiguous to the duplex is notannealed to the pilot nucleic acid, thus forming a junction site betweenthe annealed region and the non-annealed region.

Second, this cleavage structure is exposed to a cleavage agent capableof preferentially cleaving the cleavage structure at a target site. Thecleavage agent acts independently of sequence of the cleavage structure.

Third, the cleavage structure and the cleavage agent are incubated underconditions wherein cleavage can occur.

In a preferred form of the present invention, the pilot nucleic acid iscovalently linked to the target molecule. In another preferred form ofthe present invention, the pilot nucleic acid is an oligonucleotide.

In a preferred form of the present invention the cleavage entity is anenzyme. In an especially preferred form of the present invention, thecleavage agent is a domain of a DNA polymerase. In another preferredform of the present invention, the cleavage agent is thermostable.

In another preferred form of the present invention, the third step ofthe method of the present invention is at a temperature between 40° C.and 85° C.

In a preferred form of the invention, the target site is within twonucleotides of the junction site.

It is an object of the present invention to cleave nucleic acidmolecules, such as RNA and DNA molecules, at specific sites.

It is a feature of the present invention that a nucleic acid moleculemay be cleaved at any desired location in a sequence independent manner.

It is an advantage of the present invention that the cleavage can takeplace at high temperatures.

It is another advantage of the present invention that cleavage sites arenot restricted to specific restriction endonuclease sites.

Other objects, advantages, and features of the present invention willbecome apparent after examination of the specification, drawings andclaims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of a bifurcated duplex comprising a region of thetarget molecule hybridized to a complementary region of the pilotnucleic acid.

FIG. 2 is a diagram of a bifurcated duplex comprising a region of thetarget molecule, pilot oligonucleotide and a primer--a nucleic acidcomplementary to the 3' extension of the pilot nucleic acid.

FIG. 3 is a diagram of a bifurcated duplex comprising a target moleculeand pilot oligonucleotide covalently attached to a primer.

FIG. 4 is a diagram of a bifurcated duplex with no 3' extension of thepilot nucleic acid.

FIG. 5 is the nucleotide sequence of an exemplary bifurcated duplex andprimer.

FIG. 6 is a stained gel demonstrating attempts to amplify a bifurcatedduplex with DNAPTaq or DNAPStf.

FIG. 7 is an autoradiogram of a gel analyzing the cleavage of abifurcated duplex by DNAPTaq and lack of cleavage by DNAPStf.

FIG. 8 is a set of autoradiograms of gels analyzing cleavage or lack ofcleavage upon addition of different reaction components and change ofincubation temperature during attempts to cleave a bifurcated duplexwith DNAPTaq. Panel A depicts the results of cleavage assays run in thepresence or absence of DNA Taq, MgCl₂, dNTPS or primers. Panel B depictsthe results on the cleavage assays at different temperatures.

FIG. 9 is an autoradiogram displaying timed cleavage reactions, with andwithout primer. Panel A depicts reactions with or without added primer,as indicated. Panel B depicts reactions without added primer.

FIG. 10 is an autoradiogram of a gel demonstrating attempts to cleave abifurcated duplex (with and without primer) with various DNAPs. Panel Adepicts assays with Pfu, Taq, Tfl, Tth, and Tli polymerases. Panel Bdepicts assays with Taq, Ecl, Kln polymerases.

DESCRIPTION OF THE INVENTION

1. In General

The present invention is a method for cleaving a nucleic acid molecule.By "nucleic acid" we mean any poly-nucleotide. This method takenadvantage of the observation that 5' nucleases of DNA polymerases(DNAPs) recognize a substrate by structure rather than sequence. Thisfeature can be exploited to impose sequence specificity on the enzyme byannealing a targeted sequence at high temperatures to an appropriatepilot nucleic acid which directs the enzyme to cleave the substrate atthe desired site. It also can be used as an indirect probe to analyzesequence or structural differences between two similar nucleic acids orcomplexes containing nucleic acids.

FIG. 1 is a diagram of an appropriate nucleotide substrate for thepresent invention. We call such appropriate substrates "cleavagestructures". Referring to FIG. 1, the cleavage structure of the presentinvention comprises a target molecule and a pilot nucleic acid. Thetarget nucleic acid molecule has a first region which is sufficientlycomplimentary to anneal the pilot nucleic acid, called a pilot"oligonucleotide" in FIG. 1. After annealing, the 5' region of thetarget molecule of FIG. 1 is free and the 3' region of the targetmolecule is annealed. We call the FIG. 1 structure a "bifurcatedduplex." The annealed region and the non-annealed region define a"junction site". By "junction site" we mean the site that is between thefirst nucleotide in duplex form and the non-annealed nucleotides.

After the annealing reaction, the annealed molecule is exposed to acleavage agent. By "cleavage agent" we mean a molecule such as a DNAP, adomain of a DNAP, or a synthetically created protein or peptide, capableof cleaving a cleavage structure at a specific site. A preferableexample of a cleavage agent is a 5' nuclease activity of DNAP, such asDNAPTaq.

After exposing the cleavage structure to a cleavage agent, the structureand agent are incubated under conditions wherein cleavage can occur. TheExamples below disclose conditions suitable for the various specificcleavage agents used, such as DNAPTaq, DNAPTfl, DNAPTth and DNAPEcl.However, one skilled in the art of molecular biology would be able todetermine appropriate conditions for other cleavage agents.

After the incubation, the target molecule is cleaved at the cleavagesite. The exact location of the cleavage site is shown in FIGS. 1-4.When the bifurcated duplex of FIG. 1 is incubated with a cleavage agent,the cleavage site is within two nucleotides of the junction site. When aprimer (described below and in FIGS. 2 and 3) is part of the cleavagestructure, the cleavage site is within two nucleotides of the junctionsite if the primer extends to the junction site, as FIGS. 2 and 3depict. If the primer does not extend to the junction site, the cleavagesite may shift to a site within the 5' arm of the target nucleotide.

The method of the present invention requires single-stranded molecules.However, if one wishes to cleave a double-stranded molecule, one needonly denature the double-stranded molecule and then make individualpilot oligonucleotides to the desired sites of cleavage in both strands.

The pilot nucleic acid may either be covalently linked to the targetmolecule or it may be an exogenously added nucleic acid molecule, suchas an oligonucleotide. In FIG. 1, the pilot is shown as anoligonucleotide that has been added to the target molecule. However, theannealing pilot nucleic acid could be another region of the targetmolecule that is complementary to the first section of the targetmolecule.

Preferably, the cleavage agent is DNAPTaq. Our observation that the 5'nuclease of DNAPTaq is a specific endonuclease that can release long 5'extensions was unexpected since no previous studies on the 5' nucleaseactivities of DNAPs had reported products longer than a few nucleotides(Setlow, supra; Holland, supra). Also, the published work on the 5'nuclease of DNAPTaq indicated that this nuclease activity requiredconcomitant synthesis (Lawyer, supra; Holland, supra). This requirementhad been observed in reactions run in high concentrations of KCl, whichwould inhibit primer-independent cleavage, so an added primer would haveto have been extended up to the cleavage site.

Preferably, the incubation step is run at a temperature between 40° C.and 85° C. and the cleavage agent is a thermostable enzyme. Because thecleavage reaction can be run at 75° C. and above when DNAPTaq or anotherthermostable enzyme is used, the investigator can impose a high degreeof specificity on the site of cleavage by requiring that the interactionbetween the target and the pilot oligonucleotide be stable at hightemperatures. (By "thermostable" we mean an enzyme that is stable atincubation temperatures greater than 40° C. and preferably stable atincubation temperatures greater than 70° C. for a sufficient period oftime for cleavage to occur.) The high degree of sequence specificity maybe useful in selection against certain templates in PCR amplification.Likewise, this specificity may find application in the production ofnucleic acid fragments of an optimal size, such as those that are to beanalyzed by single-strand conformation polymorphism (SSCP, Orita, et al.Genomics 5:874, 1989; K. Hayaski, PCR Meth. and Appl. 1:34, 1991).

Other cleavage agents that are preferable for the present invention areDNAPs from Thermus thermophilus and thermos flavus. However, the methodof the present invention is also suitable for non-thermostable enzymes,such as DNAPEcl, and the incubating step may take place undertemperature conditions suitable for these enzymes.

2. Cleavage of Target Nucleic Acid Molecule Using a PilotOligonucleotide

In a preferred embodiment of the present invention, the pilot nucleicacid component of the cleavage structure is an oligonucleotide designedto be complementary to a stretch of sequence on the 3' side of and toone nucleotide on the 5' side of the desired cleavage site. FIG. 1describes this assembly and the specific cleavage site. If a cut in adouble-stranded molecule is required, the double-stranded molecule isdenatured and the procedure described below is performed on bothstrands, by separate pilot oligonucleotides. A preferable method ofdenaturing a double-stranded nucleic acid molecule is by heating themolecule to a temperature above the "melting temperature" of themolecule.

The basic procedure involves the partial annealing of a pilotoligonucleotide such that the 5' end of the pilot forms a duplex withthe single-stranded target molecule, and the 3' end of the pilot is notpaired to the target. This forms the bifurcated structure depicted inFIG. 1. Still referring to FIG. 1, the unpaired 5' region of the targetmolecule is termed the 5' arm and the unpaired 3' region of the pilotoligonucleotide is termed the 3' arm.

The complex formed is then exposed to a cleavage agent and the targetmolecule is cleaved, one nucleotide into the duplex. Referring to FIG.1, the site where the target molecule is cleaved is termed the "cleavagesite."

Long 5' arms might contain structures that could inhibit cleavage. Suchstructures may be removed by performing the reaction at elevatedtemperature with a thermostable 5' nuclease.

The arrangement shown in FIG. 1 has an unpaired 3' arm. This arm may beas short as one nucleotide or it may be very long (>2500 nucleotides).Cleavage of the target molecule in this type of complex is termed"primer-independent" cleavage, reflecting the absence of a primerannealed to the 3' arm. An excess of the pilot oligonucleotide would beannealed to the target DNA, at a temperature that would precludenonspecific binding. The reaction buffer would be chosen to maintain theappropriate pH for the enzyme, and would be low in salt. For example, ifthe 5' exonuclease activity of DNAPTaq is used as a cleavage agent, thebuffer is adjusted to pH 8.5 (at 20° C.) and the optimal salt is around20 mM for KCl. We have found that KCl concentrations above about 40 mMinhibit cleavage of the target molecule in the FIG. 1 structure. Thissalt optimum is lower than the 50 mM usually provided in forwardsynthesis reactions with DNAPTaq.

The annealed complex is then exposed to the cleavage agent and cleavageof the target molecule is monitored by standard methods, such as byelectrophoretic mobility of substrate and products.

This mode of cleavage is also useful for detecting single- orpoly-nucleotide sequence differences within the target nucleic acid. Todetermine if a target nucleic acid has a certain nucleotide in aspecific position, within an otherwise known sequence (e.g. a specificpoint mutation) a pilot oligonucleotide would be designed such that itwould hybridize completely to one of the sequence options, but with theother options, it would have at least one mismatched nucleotide at itsextreme 3' end. Successful cleavage would indicate that the pilot is notcompletely complementary to the target nucleic acid.

An alternative arrangement of target nucleic acid and pilotoligonucleotide is disclosed in FIG. 2. The arrangement shown in FIG. 2includes another oligonucleotide, which we call a "primer", annealed tothe 3' arm of the pilot oligonucleotide. Prefereably, the 3' end of theannealed primer is within 15 nucleotides of the junction site. Morepreferably, the 3' end of the primer is within 4 nucleotides of thejunction site. Most preferably, the 3' end of the primer is within 1nucleotide of the junction site.

Cleavage of this structure is termed "primer-directed" cleavage. We haveexperimentally determined that primer-directed cleavage occurs at agreater rate and is relatively insensitive to the salt concentration inthe reaction mixture. The complex depicted in FIG. 2 has the advantageof allowing salt suppression of primer-independent cleavage (FIG. 1).Therefore, cleavage at fortuitous secondary structures can be suppressedby inclusion of KCl at concentration equal to or greater than 50 mM.

The primer can be of any convenient sequence, provided that theprimer/pilot duplex is long enough to be stable at the temperaturechosen for the pilot/target annealing and is preferably at high enoughconcentration to saturate all of the pilot oligonucleotide 3' arms. Tocleave a target molecule at a specific site, a reaction would beassembled as described for primer-independent cleavage, above. In apreferred form of the present invention, the concentration of KCl can beincreased to 50 mM, or higher, to suppress cleavage at any structureslacking primers. After exposure to an enzyme with 5' nuclease activity,cleavage can be monitored as described above.

Another option for primer directed cleavage is depicted in FIG. 3. Inthis case, the pilot oligonucleotide has a 3' terminal hairpin that actsas an integral primer. The looped end of the hairpin may be of aspecific sequence called a tetra-loop, which confers extraordinarythermostability on the stem-loop structure (Antao et al., Nucl. AcidsRes., 19:5901, 1991). When the primer is covalently linked to the pilotnucleic acid the presence of a primer annealed to the 3' arm is not afunction of the primer concentration. Cleavage of a target molecule inthe presence of such a pilot has all of the advantages of a primerdirected cleavage, such as salt suppression of random cleavage, andrequires the addition of only one oligonucleotide.

Cleavage of an RNA target molecule can be achieved by formation of allof the complexes described above and, additionally, the one depicted inFIG. 4, in which the pilot oligonucleotide does not have a free 3' arm.This arrangement has the advantage that oligonucleotides designed forhybridization and/or reverse transcription may also be used as pilotoligonucleotides, and vice versa, if appropriate to the experiment.

Uses for RNA cleavage include creation of fragments of RNA forstructural studies or for subsequent translation in the study oftruncated proteins. In the method of the present invention, the 5'nuclease cleaves the RNA at a single, predictable site, in contrast topreviously described RNAse H, which cleaves the RNA at several sitesalong an RNA/DNA heteroduplex. The reaction is assembled as describedabove for the DNA cleavages, and is performed under the higher saltconditions usually used for the is primer-directed cleavage.

3. Cleavage of a Target Nucleic Acid with Internal Annealing

The cleavage method of the present invention can be used to cleave amolecule in which there is some internal homology so that a part of themolecule will anneal with another part of the molecule. This situationis useful to detect internal sequence differences in DNA fragmentswithout prior knowledge of the specific sequences of the variants. Thebifurcated duplex structure which identifies a nucleic acid as asubstrate for the 5' nuclease can be created by allowing a singlestranded molecule to fold upon itself. Such a folded structure couldresemble those shown in FIGS. 1-3, except that the 3'-end of the targetmolecule and the 5'-end of the pilot nucleic acid would be covalentlylinked. The conformation assumed by a single strand when it folds onitself depends upon the length, the specific sequence, the temperatureof the reaction, and the presence of salts and other solutes that eitherstabilize or disrupt the base-pairing.

Changes as small as a single nucleotide within a strand of nucleic acidup to about 300 nucleotides long can alter the folding structure of thatmolecule sufficiently to be detected by an altered mobility duringnon-denaturing gel electrophoresis (single Strand ConformationPolymorphism, SSCP), and the changes are of individual enough characterto allow discrimination among many closely related molecules. This samealteration of structure can be assessed by altered patterns of cleavageusing this method. One might use this type of analysis to examine humangenetic variation, such as allelic differences, or to analyze simplerorganisms, such as in the characterization of different isolates of ahighly varied or a rapidly mutating virus.

A specific fragment of RNA or DNA would be isolated from the organism bydirected 5' nuclease cleavage, by restriction digestion or by in vitroamplification (such as in vitro transcription for RNA, or PCR for DNA).The material would be denatured by heating, if double-stranded, and thereaction would be dilute enough to minimize immediate re-annealing ofcomplementary strands and to favor intramolecular interactions. Thereactions would be performed in a buffered solution, of pH appropriateto the enzyme used, and would be low in salt, to permit cleavage ofsubstrate structures that might adventitiously form along the DNAstrand. This material would be exposed to a cleavage agent, with thetemperature and duration of the reaction determined empirically to givea desirable number of cleavage products. Reactions performed at lowertemperatures may require longer incubations to compensate for reducedrates of cleavage. The products of the reaction would then be analyzedand compared to relevant standards and controls. The assay could beelectrophoretic separation, with visualization by autoradiography, or bytransfer to a hybridization membrane with subsequent probing.

When a characteristic cleavage pattern that reflects the structure ofthe molecule of interest has been established for a given fragment ofnucleic acid, the cleavage reaction of the present invention can be usedas an assay for changes in that structure. One kind of change, mentionedabove, comes from altering some portion of the sequence of the nucleicacid itself, as would be observed in analysis of a mutation, or of anallelic variation. Another kind of change could be induced by thebinding of a foreign entity to the nucleic acid before the cleavageanalysis. Examples of foreign entities include pieces of complementarynucleic acids (i.e. RNA or DNA probes), proteins (e.g. nucleic acidbinding proteins), or anything else that might be expected to interactwith a nucleic acid molecule of interest.

For example, to observe the effect of an oligonucleotide on thestructure of nucleic acid, the target molecule would be heat-denatured,as above, and cooled down to the reaction temperature in the presence ofan excess of a complementary oligonucleotide, all in the appropriatesalts and buffers. At reaction temperature, the cleavage agent would beadded, and allowed to interact with the nucleic acid for the length oftime predetermined on free nucleic acid. Control reactions missing theoligonucleotide or the nuclease would be performed in parallel. At theend of the incubation period, the reactions would be stopped, such as bythe addition of enough EDTA to inactivate the enzyme, and would beassayed, as above, for the cleavage patterns.

We note that the added foreign entity need not interact with thecleavage site directly. It need only interact with the target fragmentin such a way as to alter the structure or the accessibility ofpotential cleavage sites. For example, an added oligonucleotide mightsequester a region of the fragment that would otherwise contribute to orinhibit the formation of a cleavable structure. Also, such anoligonucleotide might participate in the cleavage as a pilot or primeroligonucleotide at sites within the target molecule. The onlyrequirement for a foreign entity in these types of experiments is thatit behaves in a consistent and repeatable way on a given fragment ofnucleic acid, under given reaction conditions.

4. Suitable Cleavage Agents

The present invention requires a cleavage agent that is able to cleave abifurcated duplex at the desired cleavage site. A suitable cleavageagent will be able to cleave a bifurcated duplex in asequence-independent manner. Restriction endonucleases that are onlyable to cleave at a specific sequence are not suitable cleavage agents.Particularly suitable nucleases are contained in thermostable enzymessuch as DNAPTaq, DNAPTfl and DNAPTth. Non-thermostable enzymes, such asDNAPEcl, are also suitable for the present invention. We envision thatthe gene 6 protein from bacteriophage T7 is also a suitable cleavageagent.

If one wishes to determine whether or not a particular agent is suitablefor the present invention, one can test whether the agent has theability to recognize and cleave bifurcated duplex substrates. Acandidate enzyme can be tested for structure-specific cleavage activityby exposing it to test complexes that have the structures shown in FIGS.1-4. An example of an appropriate nucleotide sequence is presented atSEQ ID NO:1 and in FIG. 5. A primer oligonucleotide, illustrated in FIG.5, complementary to the 3' arm is preferred for these tests. Thesequence of the FIG. 5 primer oligonucleotide is presented in SEQ IDNO:2. Referring to FIG. 5, cleavage at site "a" will be obtained whenthe primer is not extended to the junction site. Cleavage at site "b"will be obtained when the primer is extended to the junction site.

The complete test involves three test reactions: 1) a primer-directedcleavage (FIG. 2 and FIG. 5) is tested because it is relativelyinsensitive to variations in the salt concentration of the reaction andcan, therefore, be performed in whatever solute conditions the candidateenzyme normally requires for activity; 2) a similar primer-directedcleavage is performed in a buffer that would permit primer-independentcleavage, i.e. a low salt buffer, to demonstrate that the enzyme isviable under these conditions, and 3) a primer-independent cleavage(FIG. 1 and FIG. 5 without the addition of the primer) is performed inthe same low salt buffer.

Suitable DNA substrates can be chemically synthesized or can begenerated by standard recombinant DNA techniques. By the latter method,the hairpin portion of the molecule can be created by inserting into acloning vector duplicate copies of a short DNA segment, adjacent to eachother but in opposing orientation. The double-stranded fragmentencompassing this inverted repeat, and including enough flankingsequence to give short (about 20 nucleotides) unpaired 5' and 3' arms,can then be released from the vector by restriction enzyme digestion, orby PCR performed with an enzyme lacking a 5' exonuclease (e.g. theStoffel fragment of Amplitaq™ DNA polymerase, Vent™ DNA polymerase).

The test DNA can be labeled on either end, or internally, with either aradioisotope or with a non-isotopic tag. Whether the hairpin DNA is asynthetic single strand, or a cloned double strand, the DNA is heatedprior to use to melt all duplexes. When cooled on ice, the structuredepicted in FIG. 5 is formed. This structure is stable for sufficienttime to perform these assays.

For Test 1, described above, a detectable quantity of the test moleculeand preferably a 10 to 100-fold molar excess of primer are placed in abuffer known to be compatible with the test enzyme. For Test 2, the samequantities of molecules are placed in a solution that is the same as thebuffer above regarding pH, enzyme stabilizers (e.g. bovine serumalbumin, nonionic detergents, gelatin) and reducing agents (e.g.dithiothreitol, 2-mercaptoethanol) but that replaces any salt with 20 mMKCl. Buffers for enzymes that usually operate in the absence of salt arenot supplemented to achieve this concentration. For Test 3, the samequantity of the test molecule, but no primer, are combined under thesame buffer conditions used for Test 2.

All three test reactions are then exposed to enough of the enzyme thatthe molar ratio of enzyme to test complex is approximately 1:1. Theamount of enzyme needed can be calculated from the specific activity(units/mg) given by the manufacturer and the molecular weight. In ourhands, 0.5 units per 0.01 pmoles of target has always worked, regardlessof what the molar ratios were. The reactions are incubated at a range oftemperatures up to, but not exceeding, the temperature allowed by eitherthe enzyme stability or the complex stability, whichever is lower,probably up to 72° C. for enzymes from thermophiles, for a timesufficient to allow cleavage (10 to 60 minutes). The products of Tests1, 2 and 3 are typically resolved by denaturing polyacrylamide gelelectrophoresis, and visualized by autoradiography or by a comparablemethod appropriate to the labeling system used.

Release of the 5' arm as a discreet oligonucleotide or group ofoligonucleotides indicates successful internal cleavage.

With the DNAPTaq, Test 1 is performed in a buffer of 10 mM Tris-Cl, pH8.5 at 20° C., 1.5 mM MgCl₂ and 50 mM KCl, while in Tests 2 and 3 theKCl concentration is reduced to 20 mM. In tests 1 and 2, when 10 fmolesof the test molecule of FIG. 5 is combined with 1 pmole of the indicatedprimer, incubation for 10 minutes at 55° C. is sufficient for 0.1 unitsof the enzyme to cleave completely. In Test 3, incubation for 10 minutesat 72° C. gives complete cleavage of the same amount of this molecule,in the absence of primer. In both cases, the products of the reactionare stable during extended incubations (>60 minutes), provided that nocontaminating nucleases are present, so overdigestion is unlikely. Whenthe molecule shown in FIG. 5 is labeled at the 5' end, the released 5'fragment, 21 or 25 nucleotides long, is conveniently resolved on a 20%polyacrylamide gel (19:1 crosslinked) with 7M urea, in a buffer of 45 mMTris-borate pH 8.3, 1.4 mM EDTA.

Some enzymes or enzyme preparations may have associated or contaminatingactivities that may be functional under the cleavage conditionsdescribed above, and may interfere with 5' nuclease detection. Reactionconditions may need to be modified in consideration of these otheractivities, to avoid destruction of the substrate or other masking ofthe 5' nuclease cleavage and its products. For example, the DNApolymerase I of E. coli (DNAPEcl), in addition to its 5' nuclease andpolymerase activities, has a 3' exonuclease that can degrade DNA in a 3'to 5' direction. Consequently, when the molecule in FIG. 5 is exposed tothis polymerase under the conditions described above, the 3' exonucleasequickly removes the 3' arm, destroying the bifurcated structure requiredof a substrate for the 5' nuclease cleavage, and no cleavage isdetected. The true ability of DNAPEcl to cleave the structure can berevealed if the 3' exonuclease is inhibited by a change of conditions(e.g., change in reaction pH), mutation, or by addition of a competitorfor the activity. Addition of 100 pmoles of a single-stranded competitoroligonucleotide unrelated to the target molecule, to the cleavagereaction with DNAPEcl effectively inhibits the digestion of the 3' armof the molecule of FIG. 5, without interfering with the 5' nucleaserelease of the 5' arm. The concentration of the competitor is notcritical, but should be high enough to occupy the 3' exonuclease for theduration of the reaction.

Similar destruction of the test molecule may be caused by contaminantsin the candidate nuclease preparation. Several sets of these tests maybe necessary to determine the purity of the candidate nuclease, and tofind the window between under and over exposure of the test molecule tothe preparation being investigated.

EXAMPLES

A. 5' nuclease of DNAPTaq.

During the polymerase chain reaction (PCR) (Saiki, et al., Science239:487, 1988; Mullis et al., Methods Enzymol. 155:335, 1987) DNAPTaq isable to amplify many, but not all, DNA sequences. One sequence that wewere unable to amplify using DNAPTaq is the longer sequence shown inFIG. 5. This DNA sequence has the distinguishing character of being ableto fold on itself to form a hair-pin with two single-stranded arms,which correspond to the primers used in PCR.

To test whether this failure to amplify is due to the 5' nucleaseactivity of the enzyme, we compared the abilities of DNAPTaq and DNAPStfto amplify this DNA sequence during 30 cycles of PCR. Syntheticoligonucleotides were obtained from The Biotechnology Center at theUniversity of Wisconsin-Madison. The DNAPTaq and DNAPStf were fromPerkin Elmer-Cetus. Polymerase chain reactions comprised 1 ng of plasmidtarget DNA, 5 pmoles of each primer, 40 μM each dNTP, and 2.5 units ofDNAPTaq or DNAPStf, in a 50 μl solution of 10 mM Tris•Cl pH 8.3. TheDNAPTaq reactions included 50 mM KCl and 1.5 mM MgCl₂ while the DNAPStfreactions included 10 mM KCl and 4 mM MgCl₂. The temperature profile was95° C. for 30 sec., 55° C. for 1 min. and 72° C. for 1 min., through 30cycles. Ten percent of each reaction was analyzed by gel electrophoresisthrough 6% polyacrylamide (cross-linked 29:1) in a buffer of 45 mMTris•Borate, pH 8.3, 1.4 mM EDTA.

Our results are shown in FIG. 6. The expected product was made byDNAPStf but not by DNAPTaq. We conclude that the 5' nuclease activity ofDNAPTaq is responsible for the lack of amplification of this DNAsequence.

To test whether the 5' unpaired nucleotides in the substrate region ofthis structured DNA are removed by DNAPTaq, we compared the fate of theend-labeled 5' arm during four cycles of PCR using the same twopolymerases (FIG. 7). The hairpin template described in FIG. 5 was madeusing DNAPStf and a ³² P-5'-end-labeled primer. The 5'-end of the DNAwas released as a few large fragments by DNAPTaq but not by DNAPStf. Thesizes of these fragments (based on their mobilities) show that theycontain most or all of the unpaired 5' arm of the DNA. Thus, we learnedthat cleavage occurs at or near the base of the bifurcated duplex. Thesereleased fragments terminate with 3' OH groups, as evidenced by directsequence analysis, and the ability of the fragments to be extended byterminal deoxynucleotidyl transferase.

FIGS. 8-10 are different combinations of reaction components anddemonstrate our characterization of the present invention. Unlessotherwise specified, the cleavage reactions comprised 0.01 pmoles ofheat-denatured, end-labeled hairpin DNA (with the unlabeledcomplementary strand also present), 1 pmole primer (complementary to the3' arm) and 0.5 units of DNAPTaq (estimated 0.026 pmoles) in a totalvolume of 10 μl of 10 mM Tris•Cl, pH 8.5, 50 mM KCl, 1.5 mM MgCl₂. Asindicated, some reactions had different concentrations of KCl, and theprecise times and temperatures used in each experiment are indicated inthe individual figures and legends. The reactions that included a primerused the one shown in FIG. 5. In some instances, the primer was extendedto the junction site through the use of polymerase and appropriatedNTPs.

Reactions were initiated at the final reaction temperature by theaddition of either the MgCl₂ or enzyme. Reactions were stopped at theirincubation temperatures by the addition of 8 μl of 95% formamide with 20mM EDTA and 0.05% marker dyes. Tm calculations listed were made usingthe Oligo™ primer analysis software from National Biosciences, Inc.,Plymouth, Minn. These were determined using 0.25 μM as the DNAconcentration, at either 15 or 65 mM total salt (the 1.5 mM MgCl₂ in allreactions was given the value of 15 mM salt for these calculations).

FIG. 8 is an autoradiogram containing the results of a set ofexperiments designed to determine the effects of reaction components andconditions on the cleavage site. FIG. 8A is a determination of reactioncomponents required for cleavage. Incubation of 5'-end-labeled hairpinDNA was for 30 minutes at 55° C., with the indicated components. Theproducts were resolved by denaturing polyacrylamide gel electrophoresisand the lengths of the products, in nucleotides, are indicated. FIG. 8Bdescribes the effect of temperature on the site of cleavage in theabsence of added primer. Reactions were incubated in the absence of KClfor 10 minutes at the indicated temperatures. The lengths of theproducts, in nucleotides, are indicated.

Surprisingly, we learned that cleavage by DNAPTaq requires neither aprimer nor dNTPs (see FIG. 8A). Thus, the 5' nuclease activity can beuncoupled from polymerization. Nuclease activity requires magnesiumions, though manganese ions can be substituted without loss of activity.Neither zinc nor calcium ions support the cleavage reaction. Thereaction occurs over a broad temperature range, from 25° C. to 85° C.,with the rate of cleavage increasing at higher temperatures.

Still referring to FIG. 8, in the absence of added dNTPs the primerinfluences both the site and the rate of cleavage of the hairpin,although the primer is not elongated under these conditions. The changein the site of cleavage (FIG. 8A) apparently results from disruption ofa short duplex formed between the arms of the DNA substrate. In theabsence of primer, the sequences indicated by underlining in FIG. 5could pair, forming an extended duplex. Cleavage at the end of theextended duplex would release the 11 nucleotide fragment seen on theFIG. 8A lanes with no added primer. Addition of excess primer (FIG. 8A,lanes 3 and 4) or incubation at an elevated temperature (FIG. 8B)disrupts the short extension of the duplex and results in a longer 5'arm and, hence, longer cleavage products.

The location of the 3' end of the primer can influence the precise siteof cleavage. We learned by electrophoretic analysis that in the absenceof primer (FIG. 8B), cleavage occurs at the end of the substrate duplex(either the extended or shortened form, depending on the temperature)between the first and second base pairs. When the primer extends up tothe base of the duplex, cleavage also occurs one nucleotide into theduplex. However, when a gap of four or six nucleotides exists betweenthe 3' end of the primer and the substrate duplex, the cleavage site isshifted four to six nucleotides in the 5' direction. This shifting ofthe cleavage site in concert with the location of the 3' end of theprimer may result from extension of the substrate duplex throughformation of short, imperfect duplexes between the 5' arm and thesingle-stranded gap downstream of the primer. This explanation isconsistent with our observation that substrates with different sequencesin this region behave in nonidentical but similar fashions.

FIG. 9 describes the kinetics of cleavage in the presence (FIG. 9A) orabsence (FIG. 9B) of a primer oligonucleotide. The reactions were run at55° C. with either 50 mM KCl (FIG. 9A) or 20 mM KCl (FIG. 9B). Thereaction products were resolved by denaturing polyacrylamide gelelectrophoresis and the lengths of the products, in nucleotides, areindicated. "M", indicating a marker, is a 5' end-labeled 19-ntoligonucleotide. Under these salt conditions FIGS. 9A and B indicatethat the reaction appears to be about twenty times faster in thepresence of primer than in the absence of primer. This effect on theefficiency may be attributable to proper alignment and stabilization ofthe enzyme on the substrate.

The relative influence of primer on cleavage rates becomes much greaterwhen both reactions are run in 50 mM KCl. We have determined that in thepresence of primer, the rate of cleavage increases with KClconcentration, up to about 50 mM. However, inhibition of this reactionin the presence of primer is apparent at 100 mM and is essentiallycomplete at 150 mM KCl. In contrast, in the absence of primer the rateis enhanced by concentrations of KCl up to 20 mM, but it is reduced atconcentrations above 30 mM. At 50 mM KCl, the reaction is almostcompletely inhibited. The inhibition of cleavage by KCl in the absenceof primer is affected by temperature, being more pronounced at lowertemperatures.

Recognition of the 5' end of the arm to be cut appears to be animportant feature of substrate recognition. We have been unable tocleave substrates that lack a free 5' end, such as circular M13 DNA.Even with substrates with defined 5' arms, the rate of cleavage byDNAPTaq is influenced by the length of the arm. In the presence ofprimer and 50 mM KCl, cleavage of a 5' extension that is 28 nucleotideslong is essentially complete within 2 minutes at 55° C. In contrast,cleavages of molecules with 5' arms of 80 and 191 nucleotides are onlyabout 90% and 40% complete after 20 minutes. Incubation at highertemperatures reduces the inhibitory effects of long extensionsindicating that secondary structure in the 5' arm or a heat-labilestructure in the enzyme may inhibit the reaction. A mixing experiment,run under conditions of substrate excess, shows that the molecules withlong arms do not preferentially tie up the available enzyme innon-productive complexes. These results may indicate that the 5'nuclease domain gains access to the cleavage site at the end of thebifurcated diplex by moving down the 5' arm from one end to the other.Longer 5' arms would be expected to have more adventitious secondarystructures (particularly when KCl concentrations are high), which wouldbe likely to impede this movement.

Cleavage does not appear to be inhibited by long 3' arms of eithertarget molecule or pilot nucleic acid, at least up to 2 kilobases. Atthe other extreme, we have found that 3' arms of the pilot nucleic acidas short as one nucleotide can support cleavage in a primer-independentreaction, albeit rather inefficiently. Fully paired oligonucleotides donot elicit cleavage of DNA templates during primer extension.

The ability of DNAPTaq to cleave molecules even when the complementarystrand contains only one unpaired 3' nucleotide may be useful inoptimizing allele-specific PCR. PCR primers that have unpaired 3' endscould act as pilot oligonucleotides to direct selective cleavage ofunwanted templates during preincubation of potential template-primercomplexes with DNAPTaq in the absence of nucleoside triphosphates.

B. 5' nuclease activities of other DNAPs.

To determine whether other 5' nucleases in other DNAPs would be suitablefor the present invention, we tested an array of enzymes, several ofwhich were reported in the literature to be free of apparent 5' nucleaseactivity. These were tested on the hairpin substrate shown in FIG. 5under conditions reported to be optimal for synthesis by each enzyme.

DNAPEcl and DNAPKln were obtained from Promega Corporation; the DNAP ofPyrococcus furious (DNAPPfu, Bargseid, et al., Strategies (Stratagene,La Jolla, Calif.) 4:34, 1991) was from Stratagene, the DNAP ofThermococcus litoralis (DNAPTli, Vent ™(exo-), Perler, et al., Proc.Natl. Acad. Sci. USA 89:5577, 1992) was from New England Biolabs, theDNAP of Thermus flavus (DNAPTfl, Kaledin, et al., Biokhimiya 46:1576,1981) was from Epicentre Technologies, and the DNAP of Thermusthermophilus (DNAPTth, Carballeira, et al., Biotechniques 9:276, 1990;Myers et al., Biochemistry 30, 7661, 1991) was from U.S. Biochemicals.

0.5 units of each DNA polymerase was assayed in a 20 μl reaction, usingeither the buffers supplied by the manufacturers for theprimer-dependent reactions, or 10 mM Tris•Cl, pH 8.5, 1.5 mM MgCl₂, and20 mM KCl for primer-independent reactions. Reactions using DNAPEcl andDNAPKln were done in 5 mM Tris•Cl, pH 7.5, 5 mM MgCl₂, 0.1 mM DTT, with100 pmole of 20mer oligonucleotide and reaction mixtures were at 37° C.before the addition of enzyme.

FIG. 10 is an autoradiogram recording the results of these tests. FIG.10A demonstrates reactions of endonucleases of DNAPs of severalthermophilic bacteria. The reactions were incubated at 55° C. for 10minutes in the presence of primer or at 72° C. for 30 minutes in theabsence of primer, and the products were resolved by denaturingpolyacrylamide gel electrophoresis. The lengths of the products, innucleotides, are indicated. FIG. 10B demonstrates endonucleolyticcleavage by the 5' nuclease of DNAPEcl. The DNAPEcl and DNAPKlnreactions were incubated for 5 minutes at 37° C. Note the light band ofcleavage products of 25 and 11 nucleotides in the DNAPEcl lanes (made inthe presence and absence of primer, respectively). FIG. 6B alsodemonstrates DNAPTaq reactions in the presence (+) or absence (-) ofprimer. These reactions were run in 50 mM and 20 mM KCl respectively,and were incubated at 55° C. for 10 minutes.

Referring to FIG. 10A, DNAPs from the eubacteria Thermus thermophilusand Thermus flavus cleave the substrate at the same place as DNAPTaq,both in the presence and absence of primer. In contrast, DNAPs from thearchaebacteria Pyrococcus furiosus and Thermococcus litoralis are unableto cleave the substrates endonucleolytically. The DNAPs from Pyrococcusfurious and Thermococcus litoralis share little sequence homology witheubacterial enzymes (Ito, et al., Nucl. Acids. Res. 19:4045, 1991;Mathur, et al., Nucl. Acids. Res. 19:6952, 1991; see also Perler, etal). Referring to FIG. 10B, DNAPEcl also cleaves the substrate, but theresulting cleavage products are difficult to detect unless the 3'exonuclease is inhibited. The amino acid sequences of the 5' nucleasedomains of DNAPEcl and DNAPTaq are about 38% homologous (Gelfand,supra).

The 5' nuclease domain of DNAPTaq also shares about 19% homology withthe 5' exonuclease encoded by gene 6 of bacteriophage T7 (J. J. Dunn etal. J. Mol. Biol., 166:477, 1983). We have found that this nuclease,which is not covalently attached to a DNAP polymerization domain, isalso able to cleave DNA endonucleolytically, at a site similar oridentical to the site that is cut by the 5' nucleases described above,in the absence of added primers. The nature of this 5' exonucleaseprecludes testing in the presence of a primer; a primer duplexed to the3' arm would be a substrate for this activity.

C. Transcleavage.

A cleavage substrate may also be made from separate polynucleotides,comprising a targeted nucleic acid and a pilot oligonucleotide designedto direct the nuclease to the target through formation of a substratestructure at the desired point of cleavage. The resulting substratecomplex would contain a duplex with a 3' extension opposite the desiredsite of cleavage.

To demonstrate that cleavage could be directed by a pilotoligonucleotide, we incubated a single-stranded target DNA with DNAPTaqin the presence of two potential pilot oligonucleotides. Thetrans-cleavage reactions, where the target and pilot nucleic acids arenot covalently linked, includes 0.01 pmoles of single end-labeledsubstrate DNA, 1 unit of DNAPTaq and 5 pmoles of pilot oligonucleotidein a volume of 20 μl of the same buffers. These components were combinedduring a one minute incubation at 95° C., to denature the PCR-generateddouble-stranded substrate DNA, and the temperatures of the reactionswere then reduced to their final incubation temperatures. These twooligonucleotides, called 30-12 and 19-12, have 30 or 19 nucleotidescomplementary to the DNA, respectively, plus 12 nucleotide extensions attheir 3' ends that could form short hair-pins. Oligonucleotides 30-12and 19-12 can hybridize to regions of the DNA that are 85 and 27nucleotides from the 5' end of the targeted strand.

As predicted, incubation of 30-12 with a mixture of 5'-labeled targetDNA and DNAPTaq results in the production of a fragment of the DNA 85nucleotides long. Cleavage does not occur at 80° C., which is above thecalculated melting temperature of the duplex, although the cleavageactivity is still active at that temperature (as assayed using a morestable, long, self-complementary hairpin). At 55° C., several newcleavage products accumulate in the absence of added pilotoligonucleotide. We attribute this accumulation to adventitiousformation of duplexes between complementary sequences within the DNAtarget which could serve as substrates for cleavage. These products arenot observed when the reaction is run in the presence of 50 mM KCl,which suppresses cleavage in the absence of appropriate primers or wheneither of the pilot oligonucleotides is present.

The specificity of targeted cleavage was tested by analysis of theproducts made in response to incubation with 19-12 and 30-12 atdifferent temperatures. The calculated Tm for a duplex between 19-12 andits complement is just under 50° C. in the absence of KCl. As expected,incubation of the DNA with DNAPTaq and 19-12 results in formation of a27 nucleotide-long cleavage product at 50° C., but not at 75° C. Theyield of this product increases upon the addition of KCl, which raisesthe Tm well above 50° C., and, thus, increases the concentration ofsubstrate complexes at that temperature. Substitution of 30-12 for 19-12results in release of the 85 nucleotide product in all cases. Anon-specific oligonucleotide with no complementarity to the DNAtetra-loop at its 3' end), called 0-20, has no effect on cleavage at 50°C., either in the absence or presence of 50 mM KCl. Thus, thespecificity of cleavage reactions can be controlled by the extent ofcomplementarity to the target and the conditions of incubation.

D. Cleavage of RNA.

An RNA version of the sequence used in the trans-cleavage experimentsdiscussed above was tested for its ability to serve as a substrate inthe reaction. The RNA is cleaved at the expected place, in a reactionthat is dependent upon the presence of the pilot oligonucleotide.Strikingly, in the case of RNA cleavage, a 3' arm is not required forthe pilot oligonucleotide. It is very unlikely that this cleavage is dueto previously described RNaseH, which would be expected to cut the RNAin several places along the 30 base-pair long RNA-DNA duplex. The 5'nuclease of DNAPTaq is a structure-specific RNaseH that cleaves the RNAat a single site near the 5' end of the heteroduplexed region.

Both DNAPTaq and DNAPTth cleave RNA complexed to oligonucleotides in thepresence Mg⁺², but only DNAPTaq degrades the RNA in the presence ofMn⁺². This difference can help explain why DNAPTth can use RNA as atemplate in the presence of Mn⁺². Consequently, use of these enzymes asa reverse transcriptase requires conditions that do not lead to cleavageof the template.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 2    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 91 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: oligonucleotide    (iii) HYPOTHETICAL: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    TAATACGACTCACTATAGGGAGACCGGAATTCGAGCTCGCCCGGGCGAGCTCGAATTCCG60    TGTATTCTATAGTGTCACCTAAATCGAATTC91    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: oligonucleotide    (iii) HYPOTHETICAL: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    GAATTCGATTTAGGTGACACTATAGAA27    __________________________________________________________________________

We claim:
 1. A method of cleaving a target nucleic acid at a specifictarget site comprising the steps of:(a) selecting a target site on atarget nucleic acid, wherein the target nucleic acid has a first andsecond region, (b) creating a pilot nucleic acid with a first and secondregion, wherein the first region of the pilot nucleic acid contains asequence complementary to the sequence of the first region of the targetnucleic acid, (c) forming a cleavage structure comprising the targetnucleic acid and the pilot nucleic acid, wherein the first region of thetarget nucleic acid is annealed to the first region of the pilot nucleicacid to form a duplex, and wherein the second region of the targetnucleic acid is contiguous to and 5' of the duplex and is not annealedto the pilot nucleic acid, thereby forming a junction site between theduplex region and the non-annealed region, and wherein the second regionof the pilot nucleic acid is contiguous to and 3' of the duplex regionand is not annealed to another nucleic acid, and (d) exposing thecleavage structure of step (c) to a cleavage agent which cleaves thecleavage structure at a specific target site within the first region ofthe target nucleic acid which is annealed to the pilot nucleic acid toform a duplex, within two nucleotides of the junction site, in a mannerindependent of the sequence of the cleavage structure, wherein thecleavage agent is selected from the group consisting of a 5' nucleaseactivity of a DNA polymerase and the gene 6 product from bacteriophageT7, and (e) incubating the cleavage structure and cleavage agent whereinthe cleavage occurs.
 2. The method of claim 1 wherein the pilot nucleicacid is covalently attached to the target nucleic acid at a region notin the cleavage structure.
 3. A method of cleaving a target nucleic acidat a specific target site comprising the steps of:(a) forming a cleavagestructure comprising a target nucleic acid having a first and secondregion and a pilot nucleic acid having a first and second region,wherein the first region of the target nucleic acid is annealed to thefirst region of the pilot nucleic acid to form a duplex region, andwherein the second region of the target nucleic acid is contiguous toand 5' of the duplex region and is not annealed to the pilot nucleicacid, thereby forming a junction site between the duplex region and thenon-annealed region, and wherein the second region of the pilot nucleicacid is contiguous to and 3' of the duplex region and is not annealed toanother nucleic acid, and (b) exposing the cleavage structure of step(c) to a cleavage agent which cleaves the cleavage structure at aspecific target site within the first region of the target nucleic acidwhich is annealed to the pilot nucleic acid to form a duplex, within twonucleotides of the junction site, in a manner independent of thesequence of the cleavage structure, wherein the cleavage agent isselected from the group consisting of a 5' nuclease activity of a DNApolymerase and the gene 6 product from bacteriophage T7, and (c)incubating the cleavage structure and cleavage agent wherein thecleavage occurs.